Microorganisms for producing diamine and process for producing diamine using them

ABSTRACT

The present invention relates to a microorganism for producing diamine, in which activity of a protein having an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having 42% or higher sequence homology with SEQ ID NO: 6 is introduced or enhanced, and a method of producing diamine using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/306,758, which is a U.S. national phase application of InternationalPCT Patent Application No. PCT/KR2015/003066, which was filed on Mar.27, 2015, which claims priority to Korean Patent Application Nos.10-2014-0049871, filed Apr. 25, 2014. These applications areincorporated herein by reference in their entireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is HANO_051_01US_ST25.txt. The text file is 70 KB,was created on Jun. 4, 2018, and is being submitted electronically viaEFS-Web.

TECHNICAL FIELD

The present disclosure relates to a microorganism for producing diamineand a method of producing diamine using the same.

BACKGROUND ART

Biogenic amines (BAs) are nitrogenous compounds which are mainlyproduced by decarboxylation of amino acids or by amination andtransamination of aldehydes and ketones. These biogenic amines are lowmolecular weight compounds and synthesized in the metabolism ofmicroorganisms, plants and animals, and thus biogenic amines are knownas components frequently found in these cells. In particular, biogenicamines are polyamines such as spermidine, spermine, putrescine or1,4-butanediamine, and cadaverine.

In general, putrescine is an important raw material for production ofpolyamine nylon-4,6 which is produced by reacting putrescine with adipicacid. Putrescine is usually produced by chemical synthesis involvingconversion of propylene to acrylonitrile and to succinonitrile.

As a production method of putrescine using a microorganism, a method ofproducing putrescine at a high concentration by transformation of E.coli and Corynebacterium has been reported (International PatentPublication No. WO06/005603; International Patent Publication No.WO09/125924; Qian Z D et al., Biotechnol. Bioeng. 104: 4, 651-662, 2009;Schneider et al., Appl. Microbiol. Biotechnol. 88: 4, 859-868, 2010;Schneider et al., Appl. Microbiol. Biotechnol. 95, 169-178, 2012).Furthermore, studies have been actively conducted on putrescinetransporters in E. coli, yeast, plant and animal cells (K Igarashi,Plant Physiol. Biochem. 48: 506-512, 2010).

Meanwhile, cadaverine is a foul-smelling diamine compound produced byprotein hydrolysis during putrefaction of animal tissues. Cadaverine hasthe chemical formula of NH₂(CH₂)₅NH₂, which is similar to that ofputrescine.

Cadaverine serves as a component of polymers such as polyamide orpolyurethane, chelating agents, or other additives. In particular,polyamide having an annual global market of 3.5 million tons is known tobe prepared by polycondensation of cadaverine or succinic acid, and thuscadaverine has received much attention as an industrially usefulcompound.

Cadaverine is a diamine found in a few microorganisms (Tabor and Tabor,MicrobiolRev., 49:81-99, 1985). In the gram negative bacterium E. coli,cadaverine is biosynthesized from L-lysine by L-lysine decarboxylase.The level of cadaverine in E. coli is regulated by biosynthesis,degradation, uptake and export of cadaverine (Soksawatmaekhin et al.,MolMicrobiol., 51:1401-1412, 2004).

DISCLOSURE Technical Problem

The present inventors have made intensive efforts to investigate aprotein having an ability to export diamine such as putrescine orcadaverine so as to improve diamine productivity in a microorganismhaving the diamine productivity. As a result, they found that aArcanobacterium haemolyticum-derived protein or a protein having highamino acid sequence homology therewith has a diamine export activity,and this protein is introduced into a microorganism for producingdiamine to enhance its activity, resulting in a remarkable increase inthe ability to export diamine such as putrescine and cadaverine, therebycompleting the present invention.

Technical Solution

An object of the present invention is to provide a microorganism forproducing diamine.

Another object of the present invention is to provide a method ofproducing diamine, including the steps of (i) culturing themicroorganism for producing diamine to obtain a cell culture; and (ii)recovering diamine from the cultured microorganism or the cell culture.

BEST MODE

In an aspect to achieve the above objects, the present inventionprovides a microorganism for producing diamine, in which activity of aprotein having an amino acid sequence of SEQ ID NO: 6 or an amino acidsequence having 42% or higher sequence homology with SEQ ID NO:6 isintroduced or enhanced.

As used herein, the term “diamine” collectively refers to a compoundhaving two amine groups, and specific examples thereof may includeputrescine and cadaverine. Putrescine is tetramethylenediamine which maybe produced from ornithine as a precursor. Cadaverine is called1,5-pentanediamine or pentamethylenediamine, which may be produced fromlysine as a precursor. Such diamines are industrially applicablecompounds that serve as valuable raw materials for synthesis of polymerssuch as polyamine nylon, polyamide or polyurethane.

As used herein, the term “protein having an amino acid sequence of SEQID NO: 6” is a protein found in Arcanobacterium haemolyticum, and alsocalled ARCH_0271. It was investigated that this protein retains highhomology with a membrane protein of Corynebacterium, NCgl2522. In anembodiment of the present invention, ARCH_0271 protein is identified asa putative protein which is involved in diamine export in a strainhaving diamine productivity, thereby remarkably increasing diamineproductivity.

Here, ARCH_0271 protein having the amino acid sequence of SEQ ID NO: 6may be a protein that is encoded by a nucleotide sequence of SEQ ID NO:5 or SEQ ID NO: 7. In the polynucleotide encoding the ARCH_0271 protein,however, various modifications may be made in the coding region providedthat they do not change the amino acid sequence of the polypeptideexpressed from the coding region, due to codon degeneracy or inconsideration of the codons preferred by an organism in which theprotein is to be expressed. Thus, the CE2495 protein may be encoded byvarious nucleotide sequences as well as by the nucleotide sequence ofSEQ ID NO: 5 or SEQ ID NO: 7. Of them, the nucleotide sequencerepresented by SEQ ID NO. 7 is a sequence prepared by optimizing theARCH_271 gene (SEQ ID NO. 5) for codon usage of Corynebacteriumglutamicum, but is not limited thereto.

Further, the ARCH_0271 protein of the present invention may be anyprotein having the amino acid sequence of SEQ ID NO: 6, or having 40% orhigher, preferably 60% or higher, more preferably 80% or higher, muchmore preferably 90% or higher, even much more preferably 95% or higher,and most preferably 99% or higher homology therewith, as long as theprotein exhibits a substantial diamine export activity. It is apparentthat an amino acid sequence having such homology, of which a part isdeleted, modified, substituted, or added, is also within the scope ofthe present invention, as long as the resulting amino acid sequence hasa biological activity substantially equivalent or corresponding to theprotein of SEQ ID NO: 6.

As used herein, the term “protein having an amino acid sequence having42% or higher sequence homology with the amino acid sequence of SEQ IDNO: 6” means any protein without limitation, as long as the protein hasan amino acid sequence having 42% or higher sequence homology with theamino acid sequence of SEQ ID NO: 6 and it also has substantiallydiamine export activity. For example, the protein may be a proteinhaving an amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 26, but isnot limited thereto.

For example, the protein having the amino acid sequence of SEQ ID NO: 23is a protein found in Alcaligenes faecalis subsp. faecalis, and alsocalled QWA_00075. It was investigated that this protein retains 41%homology with a membrane protein of Corynebacterium, NCgl2522 and 42%homology with ARCH_0271 of Arcanobacterium haemolyticum. In anembodiment of the present invention, it was investigated that theQWA_00075 protein exhibits diamine export activity in a strain havingdiamine productivity, thereby remarkably increasing diamineproductivity.

The QWA_000075 protein having the amino acid sequence of SEQ ID NO: 23may be a protein that is encoded by a nucleotide sequence of SEQ ID NO:22 or 24. In the polynucleotide encoding this protein, however, variousmodifications may be made in the coding region provided that they do notchange the amino acid sequence of the polypeptide expressed from thecoding region, due to codon degeneracy or in consideration of the codonspreferred by an organism in which the protein is to be expressed. Thus,this protein may be encoded by various nucleotide sequences as well asby the nucleotide sequence of SEQ ID NO: 22 or 24. Of them, thenucleotide sequence represented by SEQ ID NO: 24 is a sequence preparedby optimizing the QWA_00075 gene (SEQ ID NO: 22) for codon usage ofCorynebacterium glutamicum, but is not limited thereto.

Further, the protein having the amino acid sequence of SEQ ID NO: 26 isa protein found in Stenotrophomonas maltophilia, and also calledSMD_2351. It was investigated that this protein retains 52% homologywith a membrane protein of Corynebacterium, NCgl2522 and 47% homologywith ARCH_0271 of Arcanobacterium haemolyticum. In an embodiment of thepresent invention, it was investigated that the SMD_2351 proteinexhibits diamine export activity in a strain having diamineproductivity, thereby remarkably increasing diamine productivity.

The SMD_2351 protein having the amino acid sequence of SEQ ID NO: 26 maybe a protein that is encoded by a nucleotide sequence of SEQ ID NO: 25or 27. In the polynucleotide encoding this protein, however, variousmodifications may be made in the coding region provided that they do notchange the amino acid sequence of the polypeptide expressed from thecoding region, due to codon degeneracy or in consideration of the codonspreferred by an organism in which the protein is to be expressed. Thus,this protein may be encoded by various nucleotide sequences as well asby the nucleotide sequence of SEQ ID NO: 25 or 27. Of them, thenucleotide sequence represented by SEQ ID NO: 27 is a sequence preparedby optimizing the SMD_2351 gene (SEQ ID NO: 25) for codon usage ofCorynebacterium glutamicum, but is not limited thereto.

The term “homology”, as used herein with regard to a sequence, refers toidentity with a given amino acid sequence or nucleotide sequence, andthe homology may be expressed as a percentage. In the present invention,a homology sequence having identical or similar activity to the givenamino acid sequence or nucleotide sequence is expressed as “% homology”.For example, homology may be identified using a standard softwareprogram which calculates parameters of score, identity and similarity,specifically BLAST 2.0, or by comparing sequences in a Southernhybridization experiment under stringent conditions as defined. Definingappropriate hybridization conditions are within the skill of the art(e.g., see Sambrook et al., 1989, infra), and determined by a methodknown to those skilled in the art.

As used herein, the term “microorganism for producing diamine” refers toa microorganism prepared by providing diamine productivity for a parentstrain having no diamine productivity or a microorganism havingendogenous diamine productivity. Specifically, the microorganism havingdiamine productivity may be a microorganism having putrescine orcadaverine productivity.

The “microorganism having putrescine productivity” may be, but is notlimited to, a microorganism in which the activity of acetylglutamatesynthase that converts glutamate to N-acetylglutamate, ornithineacetyltransferase (ArgJ) that converts acetyl ornithine to ornithine,acetylglutamate kinase (ArgB) that converts acetyl glutamate toN-acetylglutamyl phosphate, acetyl-gamma-glutamyl-phosphate reductase(ArgC) that converts acetyl glutamyl phosphate to N-acetyl glutamatesemialdehyde, or acetylornithine aminotransferase (ArgD) that convertsacetyl glutamate semialdehyde to N-acetylornithine is enhanced comparedto its endogenous activity, in order to enhance the biosynthetic pathwayfrom glutamate to ornithine, and the productivity of ornithine which isused as a precursor for putrescine biosynthesis is enhanced, but is notlimited thereto.

Further, the microorganism having putrescine productivity may be amicroorganism which is modified to have activity of ornithine carbamoyltransferase (ArgF) involved in synthesis of arginine from ornithine, aprotein (NCgl1221) involved in glutamate export, and/or a protein(NCgl469) involved in putrescine acetylation weaker than its endogenousactivity, and/or is modified to be introduced with activity of ornithinedecarboxylase (ODC).

Here, as non-limiting examples, the acetyl gamma glutamyl phosphatereductase (ArgC) may have an amino acid sequence of SEQ ID NO: 15, theacetylglutamate synthase or ornithine acetyltransferase (ArgJ) may havean amino acid sequence of SEQ ID NO: 16, the acetyl glutamate kinase(ArgB) may have an amino acid sequence of SEQ ID NO: 17, and theacetylornithine aminotransferase (ArgD) may have an amino acid sequenceof SEQ ID NO: 18. However, the amino acid sequences of respective enzymeproteins are not particularly limited thereto, and the enzymes may beproteins having amino acid sequences having 80% or higher, preferably90% or higher, or more preferably 95% or higher homology therewith, aslong as they have activities of the respective enzymes.

Further, as non-limiting examples, the ornithine carbamoyl transferase(ArgD) may have an amino acid sequence represented by SEQ ID NO: 19, theprotein involved in glutamate export may have an amino acid sequencerepresented by SEQ ID NO: 20, and ornithine decarboxylase (ODC) may havean amino acid sequence represented by SEQ ID NO: 21. However, the aminoacid sequences of respective enzyme proteins are not particularlylimited thereto, and the enzymes may include amino acid sequences having80% or higher, preferably 90% or higher, more preferably 95% or higher,or particularly preferably 97% or higher homology therewith, as long asthey have activities of the respective enzymes.

Meanwhile, the “microorganism having cadaverine productivity” may be,but is not limited to, a microorganism prepared by additionallyintroducing or enhancing activity of lysine decarboxylase (LDC) in amicroorganism having lysine productivity. For example, the microorganismmay be one having enhanced lysine productivity in order to increasecadaverine production. A method of enhancing lysine productivity may beperformed by a known method which is predictable to those skilled in theart.

The lysine decarboxylase is an enzyme catalyzing conversion of lysine tocadaverine, and its activity is introduced or enhanced, therebyeffectively producing cadaverine.

The lysine decarboxylase may have an amino acid sequence of SEQ ID NO:33, but is not particularly limited thereto. The enzyme may have anamino acid sequence having 80% or higher, preferably 90% or higher, ormore preferably 95% or higher homology therewith, as long as it has theabove activity.

As used herein, the term “production” is a concept includingextracellular release of diamine, for example, release of diamine into aculture medium, as well as production of diamine within a microorganism.

Meanwhile, the term “introduction of protein activity”, as used herein,means that a microorganism having no endogenous protein is externallyprovided with an activity of the protein, and for example, it may beperformed by introduction of a foreign gene. Further, the term“enhancement of protein activity” means that active state of the proteinretained in or introduced into the microorganism is enhanced, comparedto its intrinsic active state.

Non-limiting examples of the introduction or enhancement of the proteinactivity may include improvement of the activity of the protein itselfpresent in a microorganism due to mutation so as to achieve effectsbeyond the endogenous functions, and/or improvement in endogenous geneactivity of the protein present in the microorganism, amplification ofthe endogenous gene by internal or external factors, increase in thegene copy number, increase in the activity by additional introduction ofa foreign gene or replacement or modification of a promoter, but are notlimited thereto.

The increase in the gene copy number may be, but is not particularlylimited to, performed by operably linking the gene to a vector or byintegrating it into the host cell genome. Specifically, the copy numberof the polynucleotide in the host cell genome may be increased byintroducing into the host cell the vector which is operably linked tothe polynucleotide encoding the protein of the present invention andreplicates and functions independently of the host cell, or byintroducing into the host cell the vector which is operably linked tothe polynucleotide and is able to integrate the polynucleotide into thehost cell genome.

As used herein, “modification of the expression regulatory sequence forincreasing the polynucleotide expression” may be, but is notparticularly limited to, done by inducing a modification on theexpression regulatory sequence through deletion, insertion,non-conservative or conservative substitution of nucleotide sequence, ora combination thereof in order to further enhance the activity ofexpression regulatory sequence, or by replacing the expressionregulatory sequence with a nucleotide sequence having stronger activity.The expression regulatory sequence includes, but is not particularlylimited to, a promoter, an operator sequence, a sequence coding for aribosome-binding site, and a sequence regulating the termination oftranscription and translation.

As used herein, the replacement or modification of a promoter, althoughnot particularly limited thereto, may be performed by replacement ormodification with a stronger promoter than the original promoter. Astrong heterologous promoter instead of the original promoter may belinked upstream of the polynucleotide expression unit, and examples ofthe strong promoter may include a CJ7 promoter, a lysCP1 promoter, anEF-Tu promoter, a groEL promoter, an aceA or aceB promoter, andspecifically, a Corynebacterium-derived promoter, lysCP1 promoter or CJ7promoter is operably linked to the polynucleotide encoding the enzyme sothat its expression rate may be increased. Here, the lysCP1 promoter isa promoter improved through nucleotide sequence substitution of thepromoter region of the polynucleotide encoding aspartate kinase andaspartate semialdehyde dehydrogenase (WO 2009/096689). Further, CJ7promoter is a strong promoter derived from Corynebacterium ammoniagenes(Korean Patent No. 0620092 and WO 2006/065095).

Furthermore, modification of a polynucleotide sequence on chromosome,although not particularly limited thereto, may be performed by inducinga mutation on the expression regulatory sequence through deletion,insertion, non-conservative or conservative substitution ofpolynucleotide sequence, or a combination thereof in order to furtherenhance the activity of the polynucleotide sequence, or by replacing thesequence with a polynucleotide sequence which is modified to havestronger activity.

As used herein, the term “vector” refers to a DNA construct including anucleotide sequence encoding the desired protein, which is operablylinked to an appropriate expression regulatory sequence to express thedesired protein in a suitable host cell. The regulatory sequence mayinclude a promoter that can initiate transcription, an optional operatorsequence for regulating the transcription, a sequence encoding asuitable mRNA ribosome binding site, and a sequence regulating thetermination of transcription and translation. After the vector isintroduced into the suitable host cell, it may replicate or functionindependently of the host genome, and may be integrated into the genomeitself.

The vector used in the present invention is not particularly limited, aslong as it is able to replicate in the host cell, and any vector knownin the art may be used. Examples of conventional vectors may include anatural or recombinant plasmid, cosmid, virus and bacteriophage. Forinstance, pWE15, M13, λMBL3, λM8L4, λIXIX, λASHII, λAPII, λt10, λt11,Charon4A, and Charon21A may be used as a phage vector or cosmid vector.pBR type, pUC type, pBluescriptII type, pGEM type, pTZ type, pCL typeand pET type may be used as a plasmid vector. A vector usable in thepresent invention is not particularly limited, and any known expressionvector may be used. Preferably, pDZ, pACYC177, pACYC184, pCL, pECCG117,pUC19, pBR322, pMW118, or pCC1BAC vector may be used.

Further, the polynucleotide encoding the desired endogenous protein inthe chromosome can be replaced by a mutated polynucleotide using avector for bacterial chromosomal insertion. The insertion of thepolynucleotide into the chromosome may be performed by any method knownin the art, for example, homologous recombination. Since the vector ofthe present invention may be inserted into the chromosome by homologousrecombination, it may further include a selection marker to confirmchromosomal insertion. The selection marker is to select cells that aretransformed with the vector, that is, to confirm insertion of thedesired polynucleotide, and the selection marker may include markersproviding selectable phenotypes, such as drug resistance, auxotrophy,resistance to cytotoxic agents, or surface protein expression. Onlycells expressing the selection marker are able to survive or to showdifferent phenotypes under the environment treated with the selectiveagent, and thus the transformed cells may be selected.

As used herein, the term “transformation” means the introduction of avector including a polynucleotide encoding a target protein into a hostcell in such a way that the protein encoded by the polynucleotide isexpressed in the host cell. As long as the transformed polynucleotidecan be expressed in the host cell, it can be either integrated into andplaced in the chromosome of the host cell, or exist extrachromosomally.Further, the polynucleotide includes DNA and RNA encoding the targetprotein. The polynucleotide may be introduced in any form, as long as itcan be introduced into the host cell and expressed therein. For example,the polynucleotide may be introduced into the host cell in the form ofan expression cassette, which is a gene construct including all elementsrequired for its autonomous expression. Typically, the expressioncassette includes a promoter operably linked to the polynucleotide,transcriptional termination signals, ribosome binding sites, ortranslation termination signals. The expression cassette may be in theform of a self-replicable expression vector. Also, the polynucleotide asit is may be introduced into the host cell and operably linked tosequences required for expression in the host cell.

Further, as used herein, the term “operably linked” means a functionallinkage between a polynucleotide sequence encoding the desired proteinof the present invention and a promoter sequence which initiates andmediates transcription of the polynucleotide sequence.

Further, the microorganism having diamine productivity may be amicroorganism, in which the diamine acetyltransferase activity isweakened compared to the endogenous activity, in order to increasediamine production.

As used herein, the term “diamine acetyltransferase” is an enzymecatalyzing transfer of an acetyl group from acetyl-CoA to diamine, andit may be exemplified by Corynebacterium glutamicum NCgl1469 or E. coliSpeG, but its name may differ depending on the species of amicroorganism having diamine productivity. NCgl1469 may have an aminoacid sequence of SEQ ID NO: 12 or 13, and SpeG may have an amino acidsequence of SEQ ID NO: 14, but the sequence may differ depending on thespecies of the microorganism. The protein may have an amino acidsequence having 80% or higher, preferably 90% or higher, or morepreferably 95% or higher, or particularly preferably 97% or higherhomology therewith, as long as it has the diamine acetyltransferaseactivity.

Since the diamine acetyltransferase converts diamine to acetyl-diamine(e.g., N-Ac-putrescine or N-Ac-cadaverine), diamine productivity may beincreased by weakening its activity, compared to the endogenousactivity.

As used herein, the term “endogenous activity” refers to activity of theprotein that the original microorganism possesses in its native orundenatured state, and “modified to have weakened activity, compared tothe endogenous activity” means that activity of the protein is furtherweakened compared to the activity of the corresponding protein that theoriginal microorganism possesses in the native or undenatured state.

The weakening of the protein activity means that the protein activity isreduced, compared to a non-modified strain, or the activity iseliminated. It is possible to apply a method well known in the art tothe weakening of the protein activity.

Examples of the method may include a method of replacing the geneencoding the protein on the chromosome by a gene that is mutated toreduce the enzyme activity or to eliminate the protein activity, amethod of introducing a mutation into the expression regulatory sequenceof the gene encoding the protein on the chromosome, a method ofreplacing the expression regulatory sequence of the gene encoding theprotein by a sequence having weaker activity, a method of deleting apart or an entire of the gene encoding the protein on the chromosome, amethod of introducing antisense oligonucleotide that complementarilybinds to a transcript of the gene on the chromosome to inhibittranslation of mRNA to the protein, a method of artificially adding asequence complementary to SD sequence at upstream of SD sequence of thegene encoding the protein to form a secondary structure, therebypreventing access of the ribosomal subunits, and a reverse transcriptionengineering (RTE) method of adding a promoter for reverse transcriptionat 3′-terminus of open reading frame (ORF) of the correspondingsequence, and combinations thereof, but are not particularly limitedthereto.

In detail, a partial or full deletion of the gene encoding the proteinmay be done by introducing a vector for chromosomal insertion into amicroorganism, thereby substituting the polynucleotide encoding anendogenous target protein on chromosome with a polynucleotide having apartial deletion or a marker gene. The “partial” may vary depending onthe type of polynucleotide, but specifically refers to 1 to 300,preferably 1 to 100, and more preferably 1 to 50 nucleotides.

Meanwhile, the microorganism of the present invention is a microorganismhaving diamine productivity, and includes a prokaryotic microorganismexpressing the protein having the amino acid sequence of SEQ ID NO: 6,and examples thereof may include microorganisms belonging to Escherichiasp., Shigella sp., Citrobacter sp., Salmonella sp., Enterobacter sp.,Yersinia sp., Klebsiella sp., Erwinia sp., Corynebacterium sp.,Brevibacterium sp., Lactobacillus sp., Selenomanas sp., Vibrio sp.,Pseudomonas sp., Streptomyces sp., Arcanobacterium sp., Alcaligenes sp.or the like, but are not limited thereto. The microorganism of thepresent invention is specifically a microorganism belonging toCorynebacterium sp. or Escherichia sp., and more specifically,Corynebacterium glutamicum or Escherichia coli, but is not limitedthereto.

A specific example may be a microorganism prepared by deleting NCgl2522,which is a protein having putrescine export activity, from aCorynebacterium glutamicum ATCC13032-based putrescine-producing strainKCCM11240P (Korean Patent Publication No. 2013-0082478) and thenintroducing ARCH_0271 into the transposon gene. Therefore, thismicroorganism KCCM11240P ΔNCgl2522 Tn:P(cj7)-ARCH_0271 is designated asCC01-758, and deposited under the Budapest Treaty to the Korean CultureCenter of Microorganisms (KCCM) on Nov. 15, 2013, with Accession No.KCCM11476P.

In another aspect, the present invention provides a method of producingdiamine, comprising: (i) culturing the microorganism having putrescinediamine, in which activity of the protein having the amino acid sequenceof SEQ ID NO: 6 or 42% or higher sequence homology therewith isintroduced or enhanced, so as to obtain a cell culture; and (ii)recovering diamine from the cultured microorganism or the cell culture.

The diamine, the protein having the amino acid sequence of SEQ ID NO: 6or the protein having the amino acid sequence having 42% or highersequence homology therewith, the introduction of the protein activity,the enhancement of the protein activity, the diamine, and themicroorganism having diamine productivity are the same as describedabove.

In the method, the step of culturing the microorganism may be, althoughnot particularly limited to, preferably performed by batch culture,continuous culture, and fed-batch culture known in the art. In thisregard, the culture conditions are not particularly limited, but anoptimal pH (e.g., pH 5 to 9, preferably pH 6 to 8, and most preferablypH 6.8) may be maintained by using a basic chemical (e.g., sodiumhydroxide, potassium hydroxide or ammonia) or acidic chemical (e.g.,phosphoric acid or sulfuric acid). Also, an aerobic condition may bemaintained by adding oxygen or oxygen-containing gas mixture to a cellculture. The culture temperature may be maintained at 20 to 45° C., andpreferably at 25 to 40° C., and the cultivation may be performed forabout 10 to 160 hours.

Furthermore, a medium to be used for culture may include sugar andcarbohydrate (e.g., glucose, sucrose, lactose, fructose, maltose,molasse, starch and cellulose), oil and fat (e.g., soybean oil,sunflower seed oil, peanut oil and coconut oil), fatty acid (e.g.,palmitic acid, stearic acid and linoleic acid), alcohol (e.g., glyceroland ethanol), and organic acid (e.g., acetic acid) individually or incombination as a carbon source; nitrogen-containing organic compound(e.g., peptone, yeast extract, meat juice, malt extract, corn solution,soybean meal powder and urea), or inorganic compound (e.g., ammoniumsulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, andammonium nitrate) individually or in combination as a nitrogen source;potassium dihydrogen phosphate, dipotassium phosphate, orsodium-containing salt corresponding thereto individually or incombination as a phosphorus source; other essential growth-stimulatingsubstances including metal salts (e.g., magnesium sulfate or ironsulfate), amino acids, and vitamins. In the present invention, themedium may be used as a synonym for the culture liquid.

As used herein, the term “cell culture” is a material obtained byculturing a microorganism, and includes the medium, the microorganismcultured, and substances released from the microorganism cultured. Forexample, a nutrient supply source required for cell culture, such asminerals, amino acids, vitamins, nucleic acids and/or other componentsgenerally contained in culture medium (or culture liquid) in addition tothe carbon source, and the nitrogen source may be included. Further, adesired substance or an enzyme produced/secreted by the cells may beincluded.

Since diamine produced by culture may be secreted into the medium orremain in the cells, the cell culture may include diamine that isproduced by culturing the microorganism.

The method of recovering putrescine produced in the culturing step ofthe present invention may be carried out, for example, using a suitablemethod known in the art according to a culturing method, for example,batch culture, continuous culture, or fed-batch culture, therebycollecting the desired amino acids from the culture liquid.

Advantageous Effects

In the present invention, it is demonstrated that Arcanobacteriumhaemolyticum-derived ARCH_0271 protein is a protein having diamineexport activity, and putrescine export activity can be enhanced byintroducing this protein activity into Corynebacterium sp. microorganismwhich has a putrescine synthetic pathway, but low putrescine exportactivity. It is also demonstrated that the production of putrescine andcadaverine can be increased at the same time by introducing this proteinactivity into E. coli which has synthetic pathways of putrescine andcadaverine. Accordingly, diamine can be effectively produced by applyingArcanobacterium haemolyticum-derived ARCH_0271 protein to amicroorganism having diamine productivity.

MODE FOR INVENTION

Hereinafter, the present invention will be described in more detail withreference to Examples. However, these Examples are for illustrativepurposes only, and the invention is not intended to be limited by theseExamples.

Reference Example 1. Preparation of Corynebacterium sp. MicroorganismHaving Putrescine Productivity

It was confirmed that putrescine production was reduced when NCgl2522, apermease belonging to major facilitator superfamily (MFS), was deletedin a Corynebacterium glutamicum ATCC13032-based putrescine-producingstrain KCCM11240P (Korean Patent Publication NO. 2013-0082478) and aCorynebacterium glutamicum ATCC13869-based putrescine-producing strainDAB12-a ΔNCgl1469 (argF deletion, NCgl1221 deletion, E. coli speCintroduction, arg operon promoter substitution, NCgl1469 deletion;designated as DAB12-b, Korean Patent Publication NO. 2013-0082478) asCorynebacterium sp. microorganisms having putrescine productivity.

It was also confirmed that putrescine was produced in a high yield inCorynebacterium glutamicum strains prepared by additional introductionof NCgl2522 gene into the transposon in KCCM11240P or DAB12-b, or bysubstitution of NCgl2522 promoter on the chromosome with cj7 promoter toenhance NCgl2522 activity. Further, the intracellular amount ofputrescine was measured in the strain in which NCgl2522 expression wasenhanced, and as a result, a smaller amount of putrescine was observed,compared to that of a control group. It is indicating that NCgl2522 hasan ability to export putrescine.

In detail, based on the nucleotide sequence of the gene encodingNCgl2522 of Corynebacterium glutamicum ATCC13032, a pair of primers ofSEQ ID NOS: 1 and 2 for obtaining a homologous recombination fragment ofthe N-terminal region of NCgl2522 and a pair of primers of SEQ ID NOS: 3and 4 for obtaining a homologous recombination fragment of theC-terminal region of NCgl2522 were used as in the following Table 1.

TABLE 1 Primer Sequence (5′→3′) NCg12522-del-F1_CGGGATCCCACGCCTGTCTGGTCGC BamHI (SEQ ID NO: 1) NCg12522-del-R1_ACGCGTCGACGGATCGTAACTGTAACGAATGG SalI (SEQ ID NO: 2) NCg12522-del-F2_ACGCGTCGACCGCGTGCATCTTTGGACAC SalI (SEQ ID NO: 3) NCg12522-del-R2_CTAGTCTAGAGAGCTGCACCAGGTAGACG XbaI (SEQ ID NO: 4)

PCR was performed using the genomic DNA of Corynebacterium glutamicumATCC13032 as a template and two pairs of primers so as to amplify PCRfragments of the N-terminal and C-terminal regions, respectively. ThesePCR fragments were electrophoresed to obtain the desired fragments. Atthis time, PCR reaction was carried out for 30 cycles of denaturationfor 30 seconds at 95° C., annealing for 30 seconds at 55° C., andextension for 30 seconds at 72° C. The fragment of the N-terminal regionthus obtained was treated with restriction enzymes, BamHI and SalI, andthe fragment of the C-terminal region thus obtained was treated withrestriction enzymes. SalI and XbaI. The fragments thus treated werecloned into the pDZ vector treated with restriction enzymes, BamHI andXbaI, so as to construct a plasmid pDZ-1′NCgl2522 (K/O).

The plasmid pDZ-1′NCgl2522 (K/O) was introduced into Corynebacteriumglutamicum KCCM11240P by electroporation, so as to obtain atransformant. Then, the transformant was plated and cultured on BHISplate (37 g/l of Braine heart infusion, 91 g/l of sorbitol, and 2% agar)containing kanamycin (25 μg/ml) and X-gal(5-bromo-4-chloro-3-indolin-D-galactoside) for colony formation. Fromthe colonies thus formed, blue-colored colonies were selected as thestrain introduced with the plasmid pDZ-1′NCgl2522 (K/O).

The selected strains were cultured with shaking in CM medium (10 g/l ofglucose, 10 g/l of polypeptone, 5 g/l of yeast extract, 5 g/l of beefextract, 2.5 g/l of NaCl, and 2 g/l of urea, pH 6.6) at 30° C. for 8hours. Subsequently, each cell culture was serially diluted from 10⁻⁴ to10⁻¹⁰. Then, the diluted samples were plated and cultured on anX-gal-containing solid medium for colony formation. From the coloniesthus formed, the white colonies which appeared at relatively lowfrequency were selected to finally obtain a Corynebacterium glutamicumstrain in which the gene encoding NCgl2522 was deleted and putrescineproductivity was weakened. The Corynebacterium glutamicum strain inwhich putrescine export activity was weakened was designated asKCCM11240P ΔNCgl2522.

In the same manner, PCR was performed using the genomic DNA ofCorynebacterium glutamicum ATCC13669 as a template and two pairs ofprimers given in Table 1 so as to construct a plasmidpDZ-2′NCgl2522(K/O) by the above described method. A Corynebacteriumglutamicum strain, in which the gene encoding NCgl2522 of DAB12-b strainwas deleted using the vector according to the above described method toweaken putrescine productivity, was constructed. This Corynebacteriumglutamicum strain having weakened putrescine export activity wasdesignated as DAB12-D ΔNCgl2522.

Example 1. Selection of Arcanobacterium haemolyticum ARCH_0271

As confirmed in Reference Example 1, the NCgl2522 membrane protein wasfound to function to export putrescine. Therefore, based on the aminoacid sequence of NCgl2522, the present inventors examine genes havinghomology therewith other than genes of Corynebacterium sp. using BlastPprogram of National Center for Biotechnology Information (NCBI,www.ncbi.nlm.nih.gov), and as a result, they acquired a nucleotidesequence (SEQ ID NO: 5) and an amino acid sequence (SEQ ID NO: 6) ofARCH_0271 of Arcanobacterium haemolyticum DSM 20595, which has 56%homology therewith. Among the membrane proteins having a homology withthe amino acid sequence of NCgl2522, which are found in other speciesthan Corynebacterium sp., the amino acid sequence of ARCH_0271 isphylogenetically closest to the amino acid sequence of NCgl2522.

In the same manner, the nucleotide sequence (SEQ ID NO: 22) and aminoacid sequence (SEQ ID NO: 23) of QWA_00075 derived from Alcaligenesfaecalis subsp. faecalis NCIB 9687, which shows 41% homology with theamino acid sequence of NCgl2522, and the nucleotide sequence (SEQ ID NO:25) and amino acid sequence (SEQ ID NO: 26) of SMD_2351 derived fromStenotrophomonas maltophilia D457, which shows 52% homology with theamino acid sequence of NCgl2522, were obtained. The amino acid sequenceof QWA_00075 and the amino acid sequence of SMD_2351 show 42% and 47%homology with the amino acid sequence of ARCH_0271, respectively, asshown in the following Table 2.

TABLE 2 Comparison of amino acid sequence homology ARCH_0271 QWA_00075SMD_2351 NCgl2522 56% 41% 52% ARCH_0271 42% 47%

Meanwhile, microorganisms having genes showing homology with NCgl2522,and homology thereof are given in the following Table 3.

TABLE 3 Species Homology Acidovorax citrulli AAC00-1 47% Actinomyces sp.oral taxon 181 53% Actinomyces sp. ph3 54% Actinomyces sp. S6-Spd3 53%Actinosynnema mirum 53% Actinosynnema mirum DSM 43827 53% Alcaligenesfaecalis subsp. faecalis 41% NCIB 8687 alpha proteobacterium LLX12A 52%Arcanobacterium haemolyticum 56% Arcanobacterium haemolyticum DSM 2059556% Arsenophonus nasoniae 44% Brachybacterium paraconglomeratum 52%Brachybacterium paraconglomeratum LC44 52% Bradyrhizobium sp. BTAi1 43%Citricoccus sp. CH26A 50% Citrobacter freundii 4_7_47CFAA 53%Dermabacter hominis 1368 50% Dermabacter sp. HFH0086 51% Dietzia sp.UCD-THP 52% Enterobacteriaceae bacterium 9_2_54FAA 41% Erwinia amylovoraATCC 49946 47% Granulicoccus phenolivorans 55% Hafnia alvei ATCC 5187341% Klebsiella pneumoniae 53% Micrococcus luteus 52% Micromonospora sp.ATCC 39149 53% Mycobacterium chubuense 39% Mycobacterium gilvum 38%Mycobacterium neoaurum 39% Mycobacterium rufum 39% Nesterenkonia alba48% Nesterenkonia sp. F 51% Nocardia rhamnosiphila 40% Nocardiopsisdassonvillei 58% Nocardiopsis dassonvillei subsp. 57% dassonvillei DSM43111 Nocardiopsis kunsanensis 52% Nocardiopsis sp. CNS639 57%Nocardiopsis synnemataformans 57% Ochrobactrum anthropi ATCC 49188 46%Pectobacterium atrosepticum SCRI1043 46% Providencia alcalifaciens DSM30120 40% Pseudomonas aeruginosa 53% Pseudomonas aeruginosa C3719 46%Pseudomonas geniculata 53% Rahnella aquatilis HX2 40% Rhodococcus 40%Rhodococcus fascians 55% Rhodococcus sp. AW25M09 42% Rhodococcus sp.JVH1 40% Rhodococcus triatomae 37% Rhodococcus wratislaviensis NBRC100605 40% Salinispora 52% Salinispora arenicola 52% Salinisporapacifica 53% Salinispora tropica CNB-440 51% Sanguibacter keddieii DSM10542 52% Serratia marcescens 44% Sphingobium chinhatense 53%Stenotrophomonas maltophilia 54% Stenotrophomonas maltophilia D457 52%Stenotrophomonas sp. RIT309 52% Stenotrophomonas sp. SKA14 53%Streptococcus anginosus 52% Streptococcus anginosus CCUG 39159 51%Streptomyces 52% Streptomyces albidoflavus 54% Streptomyces alboviridis52% Streptomyces albus 55% Streptomyces albus J1074] 57% Streptomycesatroolivaceus 53% Streptomyces baarnensis 51% Streptomyces californicus52% Streptomyces cyaneofuscatus 53% Streptomyces floridae 52%Streptomyces fulvissimus 51% Streptomyces globisporus 52% Streptomycesgriseus 57% Streptomyces mediolani 55% Streptomyces sp. AA0539 48%Streptomyces sp. CcalMP-8W 52% Streptomyces sp. CNB091 55% Streptomycessp. NRRL B-1381 52% Streptomyces sp. NRRL B-3253 57% Streptomyces sp.NRRL F-2890 49% Streptomyces sp. NRRL F-5527 52% Streptomyces sp. NRRLF-5702 52% Streptomyces sp. NRRL S-623 52% Streptomyces sp. PVA 94-0757% Streptomyces sp. S4 54% Streptomyces sp. ScaeMP-e10 51% Streptomycessp. SM8 54% Streptomyces sp. W007 54% Streptomyces wadayamensis 54%Tsukamurella paurometabola 38% Turicella otitidis 56% Turicella otitidisATCC 51513 56% Xenorhabdus nematophila ATCC 19061 42% Yaniellahalotolerans 61% Yersinia enterocolitica 41%

Example 2. Fermentation of Putrescine by Introduction of Protein HavingPutrescine Export Activity into Putrescine-Producing Strain Derived fromCorynebacterium sp.

<2-1> Introduction of ARCH_0271, QWA_00075, or SMD_2351 Into TransposonGene in Chromosome of ATCC13032-Based Putrescine-Producing Strain

In order to examine whether chromosomal insertion of ARCH_0271,QWA_00075 or SMD_2351 gene affects putrescine export in KCCM11240PΔNCgl2522 having reduced putrescine export activity which was preparedin Reference Example 1, ARCH_0271, QWA_00075, or SMD_2351 was introducedinto a transposon gene by the following method.

As a vector for transformation, which allows a gene insertion into thechromosome using a transposon gene of Corynebacterium sp. microorganism,pDZTn (WO 2009/125992) was used, and cj7 (WO 2006/65095) was used as apromoter.

Based on the nucleotide sequence of Arcanobacterium haemolyticum DSM20595, ARCH_0271 gene was synthesized by optimizing the nucleotidesequence (SEQ ID NO: 7) for codon usage of Corynebacterium glutamicum.In the same manner, QWA_00075 derived from Alcaligenes faecalis subsp.faecalis NCIB 8687 and SMD_2351 derived from Stenotrophomonasmaltophilia D457 were also synthesized by optimizing the nucleotidesequences (SEQ ID NO: 24, and SEQ ID NO: 27) for codon usage ofCorynebacterium glutamicum, respectively. The genes synthesized wereobtained as genes cloned into pGEM B1.

The plasmids thus obtained were designated as pGEM B1-ARCH_0271, pGEMB1-QWA_00075, and pGEM B1-SMD_2351, respectively.

A gene fragment of about 1.51 kb, 1.53 kb, or 1.53 kb was amplifiedusing pGEM B1-ARCH_0271, pGEM B1-QWA_00075, or pGEM B1-SMD_2351 plasmidas a template and a pair of primers of SEQ ID NOs. 10 and 11, SEQ IDNOs. 28 and 29, or SEQ ID NOs. 30 and 31 (See Table 4), respectively. Atthis time, PCR reaction was carried out for 30 cycles of denaturationfor 30 seconds at 95° C., annealing for 30 seconds at 55° C., andextension for 1 minute and 30 seconds at 72° C. Next, these PCR productswere electrophoresed on a 0.8% agarose gel to elute and purify each bandof the desired size.

Further, the cj7 promoter region was obtained by carrying out PCR for 30cycles of denaturation for 30 seconds at 95° C., annealing for 30seconds at 55° C., and extension for 30 seconds at 72° C. usingp117-Pcj7-gfp as a template and a pair of primers of SEQ ID NOs. 8 and 9(See Table 4). A fragment of the cj7 promoter gene was electrophoresedon a 0.8% agarose gel to elute and purify a band of the desired size.

TABLE 4 Primer Sequence (5′→3′) CJ7-F TGTCGGGCCCACTAGT (SEQ ID NO: 8)AGAAACATCCCAGCGCTACTAATA CJ7-R AGTGTTTCCTTTCGTTGGGTACG (SEQ ID NO: 9)ARCH_0271-F CAACGAAAGGAAACACT (SEQ ID NO: 10) ATGCCAGACGTGTCCTCCARCH_0271-R GAATGAGTTCCTCGAG (SEQ ID NO: 11) TTATTCGTGTGCATATGCQWA_00075-F CAACGAAAGGAAACACT (SEQ ID NO: 28) ATGTTGCACTCCCCCACCCQWA_00075-R GAATGAGTTCCTCGAG (SEQ ID NO: 29) TTAATCAGCATGGGAGCGGCCSMD_2351-F CAACGAAAGGAAACACT (SEQ ID NO: 30) ATGCCAGCAGCGCATTCAAATAGSMD_2351-R GAATGAGTTCCTCGAG (SEQ ID NO: 31) TTAGTGCTGAGTTGGATAGGCAG

pDZTn vector was digested with XboI, and fusion cloning of each PCRproduct obtained above was performed. In-Fusion⊚HD Cloning Kit(Clontech) was used in the fusion cloning. The resulting plasmids weredesignated as pDZTn-P(cj7)-ARCH_0271, pDZTn-P(cj7)-QWA_00075, andpDZTn-P(cj7)-SMD_2351, respectively.

Each of the three plasmids pDZTn-P(cj7)-ARCH_0271,pDZTn-P(cj7)-QWA_00075, and pDZTn-P(cj7)-SMD_2351 was introduced intoCorynebacterium glutamicum KCCM11240P ΔNCgl2522 having reducedputrescine export activity described in Reference Example 1 byelectroporation to obtain transformants. The transformants were culturedwith shaking in CM medium (10 g/l of glucose, 10 g/l of polypeptone, 5g/l of yeast extract, 5 g/l of beef extract, 2.5 g/l of NaCl, and 2 g/lof urea, pH 6.8) (30° C. for 8 hours). Subsequently, each cell culturewas serially diluted from 10⁻⁴ to 10⁻¹⁰. Then, the diluted samples wereplated and cultured on an X-gal-containing solid medium for colonyformation.

From the colonies formed, the white colonies which appeared atrelatively low frequency were selected to finally obtain strains inwhich the gene encoding ARCH_0271, QWA_00075 or SMD_2351 was introducedby secondary crossover. The strains finally selected were subjected toPCR using a pair of primers of SEQ ID NOs. 8 and 11, SEQ ID NOs. 8 and29, or SEQ ID NOs. 8 and 31 to confirm introduction of the gene encodingARCH_0271, QWA_00075 or SMD_2351. These Corynebacterium glutamicummutant strain were designated as KCCM11240P ΔNCgl2522Tn:P(cj7)-ARCH_0271, KCCM11240P ΔNCgl2522 Tn:P(cj7)-QWA_00075, andKCCM11240P ΔNCgl2522 Tn:P(cj7)-SMD_2351, respectively.

<2-2> Introduction of ARCH_0271, QWA_00075, or SMD_2351 into TransposonGene in Chromosome of ATCC13869-Based Putrescine-Producing Strain

In order to examine whether the chromosomal insertion of ARCH_0271 geneaffects putrescine export in DAB12-b ΔNCgl2522 having reduced putrescineexport activity which was prepared in Reference Example 1,pDZTn-P(cj7)-ARCH_0271, pDZTn-P(cj7)-QWA_00075, or pDZTn-P(cj7)-SMD_2351prepared above was introduced into Corynebacterium glutamicum DAB12-bΔNCgl2522 and strains are confirmed introduction of ARCH_0271, QWA_00075or SMD_2351 into the transposon gene in the same manner as in Example<2-1>.

Corynebacterium glutamicum mutant strains thus selected were designatedas DAB12-b ΔNCgl2522 Tn:P(cj7)-ARCH_0271, DAB12-b ΔNCgl2522Tn:P(cj7)-QWA_00075, and DAB12-b ΔNCgl2522 Tn:P(cj7)-SMD_2351,respectively.

<2-3> Evaluation of Putrescine Productivity of Corynebacteriumsp.-Derived Putrescine-Producing Strain Introduced with ARCH_0271,QWA_00075 or SMD_2351

In order to confirm the effect of ARCH_0271 introduction on putrescineproductivity in the putrescine-producing strain, putrescineproductivities of the Corynebacterium glutamicum mutant strains preparedin Examples <2-1> and <2-2> were compared.

In detail, 10 types of Corynebacterium glutamicum mutants (KCCM11240P;KCCM11240P ΔNCgl2522; KCCM11240P ΔNCgl2522 Tn:P(cj7)-ARCH_0271;KCCM11240P ΔNCgl2522 Tn:P(cj7)-QWA_00075; KCCM11240P ΔNCgl2522Tn:P(cj7)-SMD_2351; DAB12-b; DAB12-b ΔNCgl2522; DAB12-b ΔNCgl2522Tn:P(cj7)-ARCH_0271; DAB12-b ΔNCgl2522 Tn:P(cj7)-QWA_00075, and DAB12-bΔNCgl2522 Tn:P(cj7)-SMD_2351) were plated on 1 mM arginine-containing CMplate media (1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beefextract, 0.25% NaCl, 0.2% urea, 100 μl of 50% NaOH, and 2% agar, pH 6.8,based on 1 L), and cultured at 30° C. for 24 hours, respectively. 1platinum loop of each strain thus cultured was inoculated in 25 ml oftiter medium (8% Glucose, 0.25% soybean protein, 0.50% corn steepsolids, 4% (NH₄)₂SO₄, 0.1% KH₂PO₄, 0.05% MgSO₄.7H₂O, 0.15% urea, 100 μgof biotin, 3 mg of thiamine hydrochloride, 3 mg of calcium-pantothenicacid, 3 mg of nicotinamide, and 5% CaCO₃, pH 7.0, based on 1 L), andthen cultured with shaking at 30° C. and 200 rpm for 98 hours. 1 mMarginine was added to all media for culturing the strains. Theputrescine concentration in each cell culture was measured, and theresults are shown in the following Table 5.

TABLE 5 Putrescine Strain (g/L) KCCM 11240P 12.4 KCCM 11240P ΔNCgl25221.9 KCCM 11240P ΔNCgl2522 Tn:P(cj7) - ARCH_0271 17.7 KCCM 11240PΔNCgl2522 Tn:P(cj7) - QWA_00075 4.1 KCCM 11240P ΔNCgl2522 Tn:P(cj7) -SMD_2351 3.5 DAB12-b 13.1 DAB12-b ΔNCgl2522 0.5 DAB12-b ΔNCgl2522Tn:P(cj7) - ARCH_0271 17.5 DAB12-b ΔNCgl2522 Tn:P(cj7) - QWA_00075 5DAB12-b ΔNCgl2522 Tn:P(cj7) - SMD_2351 4.1

As shown in Table 5, putrescine production was found to be increased inboth 2 types of the ARCH_0271-introduced Corynebacterium glutamicummutant strains. Further, putrescine production was found to be increasedin the QWA_00075 or SMD_2351-introduced strain, compared to the parentstrain, KCCM 11240P ΔNCgl2522 or DAB12-b ΔNCgl2522. It is indicatingthat QWA_00075 or SMD_2351 has putrescine export activity.

Example 3. Fermentation of Cadaverine by ARCH_0271 Introduction andLysine Decarboxylase Expression in Corynebacterium sp.-DerivedLysine-Producing Strain

<3-1> Introduction of ARCH_0271 into Transposon Gene in Chromosome ofL-Lysine-Producing Corynebacterium glutamicum KCCM11016P

In order to confirm cadaverine export activity of ARCH_0271 protein,ARCH_0271 gene was introduced into the chromosome of a lysine-producingstrain KCCM11016P (this microorganism was deposited at the KoreanCulture Center of Microorganisms on Dec. 18, 1995 with Accession Mo.KFCC10881, and then deposited at the International Depository Authorityunder Budapest Treaty with Accession Mo. KCCM11016P, Korean Patent No.10-0159812). pDZTn-P(cj7)-ARCH_0271 prepared above was introduced intoCorynebacterium glutamicum KCCM11016P and strain is confirmedintroduction of ARCH_0271 into transposon in the same manner as inExample <2-1>.

A Corynebacterium glutamicum mutant strain thus selected was designatedas KCCM11016P Tn:P(cj7)-ARCH_0271.

<3-2> Introduction of E. coli-Derived Lysine Decarboxylase Gene intoL-Lysine-Producing Strain Introduced ARCH_0271

The L-lysine-producing strain introduced ARCH_0271, KCCM11016PTn:P(cj7)-ARCH_0271 which was prepared in Example <3-1> was introducedwith E. coli-derived lysine decarboxylase gene in a plasmid form forcadaverine production. The nucleotide sequence (SEQ ID NO: 32) and aminoacid sequence (SEQ ID NO: 33) of lysine decarboxylase ldcC from E. coliwere obtained from NCBI data base.

An ldcC gene fragment of about 2.1 kb was obtained by carrying out PCRfor 30 cycles of denaturation for 30 seconds at 95° C., annealing for 30seconds at 52° C., and extension for 2 minutes at 72° C. using thechromosome of E. coli W3110 strain as a template and a pair of primersof SEQ ID NOS: 36 and 37 (See Table 6). This product was treated withHindIII and XbaI, and then electrophoresed in a 0.8% agarose gel toelute and purify a band of the desired size.

Further, the cj7 promoter region was obtained by carrying out PCR for 30cycles of denaturation for 30 seconds at 95° C., annealing for 30seconds at 55° C., and extension for 30 seconds at 72° C. usingp117-Pcj7-gfp as a template and a pair of primers of SEQ ID NOs. 34 and35 (See Table 6). A gene fragment of the cj7 promoter gene was treatedwith KpnI and HindII, and then electrophoresed on a 0.8% agarose gel toelute and purify a band of the desired size.

TABLE 6 Primer for promoter cj7 gene CJ7-F_KpnI CGGGGTACC(SEQ ID NO: 34) AGAAACATCCCAGCGCTACTAATA CJ7-R-HindIII CCCAAGCTT(SEQ ID NO: 35) AGTGTTTCCTTTCGTTGGGTACG Primer for E. coli ldcC geneldcC-F_HindIII CCCAAGCTT AAGCTT (SEQ ID NO: 36)ATGAACATCATTGCCATTATGGG (52) ldcC-R_XbaI TGCTCTAGA (SEQ ID NO: 37)TTATCCCGCCATTTTTAGGACTC (53)

A gene fragment which was obtained by performing electrophoresis of KpnIand XbaI-treated pECCG117 (Biotechnology letters vol 13, No. 10, p.721-726 (1991)) vector in a 0.8% agarose gel and then eluting andpurifying a band of the desired size, the cj7 promoter gene fragmenttreated with KpnI and HindIII, and the lysine decarboxylase ldcC genefragment treated with HindIII and XbaI were cloned using T4 DNA ligase(NEB). The E. coli ldcC-encoding plasmid obtained by the aboveexperiment was designated as pECCG117-Pcj7-ldcC.

The prepared pECCG117-Pcj7-ldcC vector or pECCG117 vector was introducedinto KCCM11016P and KCCM11016P Tn:P(cj7)-ARCH_0271 by electroporation,respectively. The transformants were plated on BHIS plate containing 25μg/ml kanamycin for selection. The selected strains were designated asKCCM11016P pECCG117, KCCM11016P pECCG117-Pcj7-ldcC, KCCM11016PTn:P(cj7)-ARCH_0271 pECCG117, and KCCM11016P Tn:P(cj7)-ARCH_0271pECCG117-Pcj7-ldcC, respectively.

<3-3> Evaluation of Cadaverine Productivity of Corynebacteriumsp.-Derived Lysine-Producing Strain Having Chromosomal Insertion ofARCH_0271 and Lysine Decarboxylase Gene as Plasmid

In order to examine whether introduction of ARCH_0271 into thecadaverine-producing strain affects cadaverine production, cadaverineproductivity was compared between Corynebacterium glutamicum mutantstrains prepared in Example <3-2>.

In detail, 4 types of Corynebacterium glutamicum mutant strains(KCCM11016P pECCG117; KCCM11016P pECCG117-Pcj7-ldcC; KCCM11016PTn:P(cj7)-ARCH_0271 pECCG117; and KCCM11016P Tn:P(cj7)-ARCH_0271pECCG117-Pcj7-ldcC) were cultured by the following method, andcadaverine productivity was compared therebetween.

The respective mutant strains were plated on CM plate media (1% glucose,1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2%urea, 100 μl of 50% NaOH, and 2% agar, pH 6.8, based on 1 L), andcultured at 30° C. for 24 hours. Each of the strains cultured wasinoculated to a 250 ml corner-baffled flask containing 25 ml of seedmedium (2% glucose, 1% peptone. 0.5% yeast extract, 0.15% urea, 0.4%KH₂PO₄, 0.8% K₂HPO₄, 0.05% MgSO₄ 7H₂O, 100 μg of biotin, 1000 μg ofthiamine HCl, 2000 μg of calcium-pantothenic acid, and 2000 μg ofnicotinamide, pH 7.0, based on 1 L), and cultured with shaking at 30° C.and 200 rpm for 20 hours.

Then, 1 ml of the seed culture was inoculated to a 250 ml corner-baffledflask containing 24 ml of production medium (4% Glucose, 2% (NH₄)₂SO₄,2.5% soybean protein, 5% corn steep solids 0.3% urea, 0.1% KH₂PO₄, 0.05%MgSO₄ 7H₂O, 100 μg of biotin, 1000 μg of thiamine hydrochloride, 2000 μgof calcium-pantothenic acid, 3000 μg of nicotinamide, 0.2 g of leucine,0.1 g of threonine, 0.1 g of methionine, and 5% CaCO₃, pH 7.0, based on1 L), and then cultured with shaking at 30° C. and 200 rpm for 72 hours.

After culture, cadaverine productivities were measured by HPLC. Theconcentrations of cadaverine in the cell culture of each strain aregiven in the following Table 7.

TABLE 7 Cadaverine Strain Plasmid (g/L) KCCM11016P pECCG117 0pECCG117-Pcj7-ldcC 2.3 KCCM11016P pECCG117 0 Tn:P(cj7)-ARCH_0271pECCG117-Pcj7-ldcC 2.7

As shown in Table 7, cadaverine production was increased in theARCH_0271-introduced Corynebacterium glutamicum mutant strains by morethan 17%.

Example 4. Fermentation of Diamine by Introduction of Protein HavingDiamine Export Activity into E. coli

<4-1> Preparation of Strain by Introduction of ARCH_0271, QWA_00075, orSMD_2351 into W3110

In order to examine whether expression of Arcanobacterium haemolyticumDSM 20595-derived ARCH_0271, Alcaligenes faecalis-derived QWA_00075, orStenotrophomonas maltophilia-derived SMD_2351 increases putrescine andcadaverine productions in wild-type E. coli strain W3110 havingbiosynthetic pathway of putrescine and cadaverine, Corynebacterium andE. coli shuttle vector-based pDZTn-P(cj7)-ARCH_0271,pDZTn-P(cj7)-QWA_00075, or pDZTn-P(cj7)-SMD_2351 was introduced intoW3110, respectively.

A 2×TSS solution (Epicentre) was used for transformation into E. coli,and the transformant was plated and cultured on LB plate (10 g ofTryptone, 5 g of Yeast extract, 10 g of NaCl, and 2% agar, based on 1 L)containing kanamycin (50 μg/ml) for colony formation. The colonies thusformed were designated as W3110 pDZTn-P(cj7)-ARCH_0271, W3110pDZTn-P(cj7)-QWA_00075, and W3110 pDZTn-P(cj7)-SMD_2351, respectively.

<4-2> Comparison of Diamine Productivity of E. coli Introduced withARCH_0271, QWA_00075, or SMD_2351

Putrescine and cadaverine productivities of the strains obtained abovewere examined.

In detail, W3110 and W3110 pDZTn-P(cj7)-ARCH_0271, W3110pDZTn-P(cj7)-QWA_00075, or W3110 pDZTn-P(cj7)-SMD_2351 were cultured onLB solid media at 37° C. for 24 hours.

Then, each of them was cultured in 25 ml of titration medium (2 g of(NH₄)₂PO₄, 6.75 g of KH₂PO₄, 0.85 g of citric acid, 0.7 g of MgSO₄.7H₂O,0.5% (v/v) trace element, 10 g of glucose, 3 g of AMS, and 30 g ofCaCO₃, based on 1 L) at 37° C. for 24 hours. A trace metal solutioncontained 5 M HCl: 10 g of FeSO₄.7H₂O, 2.25 g of ZnSO₄.7H₂O, 1 g ofCuSO₄.5H₂O, 0.5 g of MnSO₄. 5H₂O, 0.23 g of Na₂B₄O₇. 10H₂O, 2 g ofCaCl₂.2H₂O, and 0.1 g of (NH₄)₆Mo₇O₂.4H₂O per 1 liter.

The concentrations of putrescine and cadaverine produced from each cellculture were measured, and the results are given in the following Table8.

TABLE 8 Parent Putrescine Cadaverine strain Plasmid (mg/L) (mg/L) W3110(—) 13 5 pDZTn-P(cj7) - 51 23 ARCH_0271 pDZTn-P(cj7) - 72 30 QWA_00075pDZTn-P(cj7) - 37 15 SMD_2351

As shown in Table 8, compared to the parent strain W3110, putrescine andcadaverine productions were remarkably increased in W3110pDZTn-P(cj7)-ARCH_0271, W3110 pDZTn-P(cj7)-QWA_00075, or W3110pDZTn-P(cj7)-SMD_2351 strain which was introduced with ARCH_0271,QWA_00075 or SMD_2351, respectively.

That is, it was confirmed that the amount of diamine produced in cellculture was remarkably increased by enhancing activity of the ARCH_0271,QWA_00075 or SMD_2351 protein having 56%, 41%, or 52% sequence homologywith the amino acid sequence of NCgl2522, suggesting that the ability toexport diamine such as putrescine and cadaverine can be improved byenhancing activity of CE2495 or the protein having 42% or highersequence homology therewith.

As such, the present inventors demonstrated that Corynebacteriumglutamicum having enhanced ARCH_0271 activity prepared by introducingARCH_0271 into transposon of Corynebacterium sp. microorganismKCCM11240P ΔNCgl2522 which has a putrescine synthetic pathway, but areduced putrescine export activity has enhanced putrescine exportactivity, thereby producing putrescine in a high yield.

Accordingly, this strain KCCM11240P ΔNCgl2522 Tn:P(cj7)-ARCH_0271 wasdesignated as CC01-0758, and deposited under the Budapest Treaty to theKorean Culture Center of Microorganisms (KCCM) on Nov. 15, 2013, underAccession No. KCCM11476P.

The invention claimed is:
 1. A microorganism for producing cadaverine,wherein an activity of a protein comprising an amino acid sequencehaving at least 95% identity to SEQ ID NO:23, or SEQ ID NO:26 isintroduced or enhanced compared to the endogenous activity.
 2. Themicroorganism according to claim 1, wherein an activity of diamineacetyltransferase is further weakened compared to the endogenousactivity.
 3. The microorganism according to claim 2, wherein the diamineacetyltransferase has the amino acid sequence selected from the groupconsisting of SEQ ID NOS: 12, 13, and
 14. 4. The microorganism accordingto claim 1, wherein the microorganism is a microorganism belonging togenus Corynebacterium or genus Escherichia.
 5. The microorganismaccording to claim 4, wherein the microorganism is a microorganismbelonging to genus Corynebacterium to which an activity of lysinedecarboxylase is further introduced.
 6. A method of producingcadaverine, comprising: (i) culturing the microorganism of claim 1 toobtain a cell culture; and (ii) recovering cadaverine from the culturedmicroorganism or the cell culture.
 7. The method according to claim 6,wherein in the microorganism, an activity of diamine acetyltransferaseis further weakened compared to the endogenous activity.
 8. The methodaccording to claim 7, wherein the diamine acetyltransferase has theamino acid sequence selected from the group consisting of SEQ ID NOS:12, 13, and
 14. 9. The method according to claim 6, wherein themicroorganism is a microorganism belonging to genus Corynebacterium orgenus Escherichia.
 10. The method according to claim 9, wherein themicroorganism is a microorganism belonging to genus Corynebacterium towhich an activity of lysine decarboxylase is further introduced.